The energy-linked inner mitochondrial membrane enzyme nicotinamide nucleotide transhydrogenase (TH) couples the proton motive force (pmf) generated by respiration to formation of NADPH. In the absence of NADPH reactive oxygen species (ROS) lead to mitochondrial dysfunction which is strongly correlated with neurodegenerative diseases. In pancreatic beta cells, oxidative stress arising from TH mutations is correlated with type 2 diabetes due to loss of the redox signaling that controls insulin secretion. To understand the relationship between ROS, mitochondrial dysfunction and disease, it is necessary to understand the mechanism by which TH couples the pmf with formation of NADPH. Extensive biochemical and genetic data are available, but to define a mechanism crystal structures of the entire enzyme and its constituent subunits are required. Further, knowledge of the dispositions of the subunits during hydride transfer and proton pumping are required. A viable strategy employs TH from Thermus thermophilus for crystallization of the enzyme and its individual components. Diffraction quality crystals of the membrane intercalated domains have been obtained in the lipidic cubic phase (LCP); structures of the membrane domains in complex with the soluble, NADP(H) binding domain (domain III) will also be obtained via construct design and co- crystallization. High resolution structures of the soluble NAD(H) subunit alone and in complex with domain III have been solved. Large prismatic crystals of intact TH in multiple morphologies are available for diffraction experiments. The component crystal structures will be positioned within the TH electron density envelop using lower resolution X-ray data and EM methods (single particle averaging and electron diffraction), revealing for the first time a complete structure of TH. SAXS experiments in the presence of NAD(H) and NADP(H), together with the crystal structures, will identify motions of domain III within the complex. Structure guided site- directed mutagenesis and biochemical assays (cyclic and reverse hydride transfer, vectorial proton pumping) will define residue functions. Knowledge of structure, conformational states, and function will enable a mechanism for coupling proton translocation and NADPH formation to be deduced.